Blood product, a method of producing the same and a method of determining the virus inactivation capacity of an inactivation treatment

ABSTRACT

A blood product, exclusive of albumin, inactivated relative to infectious agents, the blood product conforming to a total virus reduction factor of at least 40, having a biological acitity of at least 50%, based on its activity prior to effecting inactivation of the infectious agents, the blood product being producible from conventional blood products and being virus-safe.

This application is a continuation of application Ser. No. 07/900,164,filed Jun. 17, 1992, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention.

The invention relates to a virus-inactivated blood product, a method ofproducing the same as well as a method of determining thevirus-inactivating capacity of an inactivation treatment.

2. Description of Related Art

By blood products, products from human or animal blood or plasma aremeant, which are intended for therapeutic, prophylactic or diagnosticapplications. Such products may contain enzymes, proenzymes includingcoagulation factors, enzyme co-factors, enzyme inhibitors,immunoglobulins, albumin, plasminogen, fibrinogen, fibronectin orplasma.

The administration of blood products involves the risk of infection dueto infectious agents possibly present in the donor plasma, such ashepatitis or AIDS viruses. Even if exclusively plasma that has beentested for its absence of these infectious agents is used, the dangerthat a patient might be infected cannot be excluded because of thelimited sensitivity of the test methods. When producing blood productsone is therefore forced to inactivate possibly present infectious agentsby various methods.

There exists a great number of publications concerned with theinactivation of pathogens in blood products.

The various methods include:

heating the blood products in aqueous solution, possibly with theaddition of virucidal substances,

heating the blood products in aqueous solution in the presence ofstabilizing agents

treating the blood products with organic solvents and/or detergents,

heating the blood products in dry and wet states,

combined treatment of the blood products with an organicsolvent/detergent and heating the blood products in the dry state.

All these inactivation methods aim at eliminating the potentialinfectiousness of the preparations, while maintaining their biologicalactivity as far as possible. However, so far this aim could only beachieved in the case of albumin preparations, by heating aqueous albuminsolutions at a temperature of 60° C. for 10 h, because albumin issubstantially more stable with regard to the influence of heat than allthe other blood proteins.

In detail, the following publications may, e.g., be mentioned to theprior art:

DE-A- 29 16 711 describes a method of treatingcoagulation-factors-containing preparations in aqueous solution byapplying a temperature of from 30° to 100° C., wherein an amino acid ora mono-, oligosaccharide or sugar alcohol are admixed to the solution ofthe coagulation factors.

EP-A2-0 053 338 describes a method of inactivating hepatitis viruses inpreparations containing factors IX and X, wherein the aqueous solutionof a blood preparation is heated in the presence of calcium ions and,possibly, an amino acid and/or a saccharide or a sugar alcohol attemperatures of up to 100° C.

In EP-A2-0 035 204 a method of inactivating aqueous protein solutions,which may contain factor VIII, fibronectin, globulin, fibrinogen andother proteins, is disclosed, wherein the composition is mixed with apolyol and the mixture is heated to a temperature of from 60° to 75° C.

In EP-A2-0 052 827 a method of inactivating hepatitis viruses in anaqueous solution containing factors II and VII in the presence of achelating agent and, possibly, an amino acid and/or a saccharide orsugar alcohol is described.

In U.S. Pat. No. 4,379,085 a method for the thermal inactivation of aplasma protein, such as C₁ -inhibitor or factor IX, in aqueous solutionin the presence of potassium or ammonium citrate is disclosed.

In EP-A2-0 077 870 an inactivation method is described, in which anaqueous, factor VIII-containing solution is heated with amino acids,monosaccharides, oligosaccharides, sugar alcohols and hydrocarbon- orhydroxy-hydrocarbon-carboxylic acids having from 3 to 10 carbon atoms,to a temperature of from 50° to 80° C.

In the PCT application WO 83/04371 a method of inactivatinghepatitis-viruses is disclosed, wherein a preparation containing thevirus is treated at a temperature of from 4° to 40° C. with ahalohydrocarbon, in particular chloroform.

EP-B1-0 015 055 discloses a method of treating a blood product, whereinthe product is subjected to a microwave radiation treatment in theanhydrous condition so as to inactivate any microorganisms present.

In a treatise of the XII. International Congress on Blood Transfusion,Abstracts, "MIR" Publishers, Moscow 1969, pp. 473-475, Rosenberg et al.disclose a method of inactivating albumin-containing preparations andfibrinogen in the dry state by heating for 10 hours at 60° C.

EP-A2-0 094 611 discloses a method of treating a factor VIII-containingcomposition in the dry, for instance the lyophilized, state by applyinga temperature of at least 60° C. for inactivating any hepatitis virusespresent.

The published PCT application WO 82/03871 describes a method of treatingpreparations containing blood coagulation enzymes, the preparationsbeing heated in the dry state so as to inactivate infectious virusespresent; the dry state is defined to be a state with less than 5% byweight of water.

Prince et al., Eur. J. of Epidemiology, Vol. 3, No. 2, Jun. 1987,discloses that a method of dry-heating a lyophilized factor VIIIconcentrate at 60°°C. for 10 h results in a limited virus inactivation,but that hepatitis and also AIDS viruses can be transmitted byadministering these dry-heated products. To increase the effectivenessof dry-heating, PCT application WO 88/08710 proposes a sequence of heattreatments.

Likewise, in EP-A-0 378 208, protein-containing compositons aresubjected to a treatment with trialkyl phosphate in combination with adry heating treatment.

The method of EP-B-0 159 311 suggests a treatment of blood products inthe solid, wet state. A content of water, methanol or ethanol of morethan 0.05 (5% by weight) and less than 0.70 (70% by weight) is adjustedand heating is effected in a closed container at a temperature in therange of from 50 ° to 121° C.

EP-B-0 050 061 discloses a method comprising the treatment of biologicaland pharmaceutical products with from 0.25 to 10% by weight of a nondenaturating amphiphile (detergent). In EP-B-0 131 740 it is, however,shown that the treatment with a detergent alone is relativelyineffective with regard to the virus inactivation. That publicationsuggests a mixture of a detergent and a di- or trialkylphosphate for aneffective treatment. In that instance, the concentration of thedetergent was 1% by weight and that of the solvent was 0.1% by weight .

It is to be understood that the use of the percentage symbol "%" refersto a percentage by weight.

A combined treatment with an organic solvent/detergent and with heat,wherein the blood product is heated in the dry state, has also beendocumented in the literature (American Journal of Hematology 35, 142(1990).

Today, virus inactivation methods are termed as effective, if afterapplication of the method on a blood product sample admixed with a highdose of a test virus (e.g., corresponding to a maximum possible titer ofapproximately 10⁵ in a coagulation factor preparation), it is no longerpossible to detect any viruses, the virus titer thus having been reducedto below the detection limit.

As a measure of inactivation, the so-called reduction factor is known,which is calculated after a single addition of the test virus from thedecadic logarithm of the quotient of the initial and final virus titers.From the directive EC III/8115/89-EN of the Commission of the EuropeanCommunities, the so-called "total reduction factor" is known. It iscalculated from the sum of the reduction factors of individualsubsequent inactivation measures.

In modern medicine it is necessary to administer many blood productsover long periods of time--in many cases even as a permanenttreatment--in large quantities, also for prophylactic reasons. This willnecessarily lead to a cumulation of infectious particles and thus to asubstantially increased risk of infection, even if preparations thathave already been virus-inactivated are administered.

Reduction factors must be compared with the so-called "worst casesituation" for a virus contamination of the entire plasma pool. Forexample, it is known that plasma that tests negative for HIV content mayyield an HIV content of up to 10⁵ ID/ml (infectious units permilliliter) in the plasma product. See Zeitschrift fur AllgemeineMedizin 65: 429-433 (1989). Thus, in the worst case, a seeminglyvirus-negative plasma product must be assumed to contain up to 10⁵ID/ml. Accordingly, virus inactivation must take into account the worstcase potential of virus contamination. For example, assuming that apatient is administered 100 l of a factor VIII preparation in the courseof his life, a method of virus-inactivating plasma derivatives must thuspermit a virus titer reduction of at least 10¹⁰ so as to avoid theinfection of a patient with AIDS viruses.

SUMMARY OF THE INVENTION

The invention has as its object to provide a virus-safe blood product,exclusive of albumin, from which it can be expected that thetransmission of infectious agents is excluded even if large quantititesof blood product are administered, and which nevertheless still has ahigh biological activity.

According to the invention, this object is achieved by a blood product,exclusive of albumin, inactivated relative to infectious agents, whichblood product conforms to a total virus reduction factor of at least 40,and has a biological activity of at least 50%, based on the activityprior to carrying out the inactivation of the infectious agents, to beobtained by an inactivation treatment in which

a) the blood product is treated in an aqueous solution containing atleast 2%, preferably at least 5%, of a detergent, and subsequently isheated in the solid state, or

b) the blood product is heated in the solid state and subsequently istreated in an aqueous solution containing at least 2%, preferably atleast 5%, of a detergent.

Preferably, the invention consists in a blood product inactivatedrelative to infectious agents, wherein the blood product is treated inan aqueous solution containing more than 10% of a detergent and isheated in the solid state.

Another embodiment of the present invention includes a blood product,exclusive of albumin, inactivated relative to infectious agents, whichblood product conforms to a total virus reduction factor of at least 40,and has a biologic activity of at least 50%, based on the activity priorto carrying out the inactivation of the infectious agents, to beobtained by an inactivation treatment comprising two or more differentinactivation methods, wherein at least one method consists in a heattreatment of the blood product in solid state having a water content offrom 5% by weight to 70% by weight. In this embodiment, advantageouslyat least one method is to be a treatment with an aqueous detergentsolution.

The biological activity is determined as the enzymatic activity (e.g.,of blood clotting enzymes and co-factors), as avidity (ofimmunoglobulins) or as antigenic activity--possibly by the use ofactivity or antigen markers.

DETAILED DESCRIPTION OF THE INVENTION

The blood product according to the invention may be produced fromconventional blood products by carrying out the inactivation treatmentfor a period of time which suffices to obtain a total virus reductionfactor of at least 40. This period of time may be determinedexperimentally in a blood product sample by repeatedly adding certainamounts of test virus during the treatment, each repetition beingeffected only when the virus titer has decreased to a certain value,preferably to below the detection limit. The total virus reductionfactor results from the sum of the individual reduction factors. Itshould be noted that detection limits can be influenced by variablessuch as the types and concentration of the sample, the type andconcentration of the detergent, and the type of contaminating virus.

Thus, if test virus is added repeatedly at chosen time intervals to abiological product during the treatment for virus inactivation, afterdetermination of the initial and final virus titer, the decadiclogarithm of the virus titer ratio may be multiplied by the number ofintervals and the reduction factors may be added up to a total virusreduction factor. This calculation requires that the virus titerreduction after the final test virus addition does not exceed theprevious titer reductions, as shown in the examples.

As the test virus, e.g., the AIDS virus or the Sindbis virus (as themodel virus for hepatitis viruses) may be used.

The invention is based on the finding that a treatment with Tween or adetergent according to the prior art which is carried out at detergentconcentrations of below 10%, does not yield a satisfactory result, ifthe blood product is not subjected to a further method of virusinactivation. This may be due to a protective effect of proteins onviruses against inactivating agents, such as detergents. This protectiveeffect may, however, be eliminated by a higher concentration of Tween ordetergent, respectively, without substantially impairing the biologicalactivity of the proteins. Such a procedure makes it possible to dowithout the addition of further substances, such as, e.g., solvents,whose toxic effect is known.

It has proven to be advantageous if the treatment with Tween ordetergent is carried out at a concentration of more than 10% and lessthan 25% by mass, for a period of time of between 1 min and 30 min, inparticular at a pH of between 5.5 and 8, at temperatures of between 0°C. and 56° C., advantageously between 15° C. and 37° C., and optionallyat an electric conductivity of from 7 to 20 mS.

A preferred method of producing inactivated blood products according tothe invention consists in that the blood product is treated with hotvapor before or after the treatment with the aqueous detergent solution,wherein the blood product is adjusted to a content of water, methanol orethanol of more than 0.05 (5% by mass) and less than 0.70 (70% by mass),preferably less than 0.40 (40% by mass) in the solid state, and istreated in a closed container at a temperature in the range of from 50°to 121° C.

The invention also relates to a method of determining the virusinactivating capacity of an inactivation treatment comprising at leastone inactivation method, by determining the reduction factor by means ofa test virus, which is characterized in that the test virus isrepeatedly added during the at least one inactivation method, and theindividual reduction factors of at least one inactivation method areoptionally added up with the reduction factors of further inactivationmethods to give a total virus reduction factor.

The invention will be explained in more detail by the followingExamples.

EXAMPLES 1

From human plasma a coagulation-factor-VIII-containing cryoprecipitatesolution was produced according to a method disclosed in AT-B 391,808.The solution was adjusted to 8% Tween 80 and 7 times admixed with aHIV-1 virus suspension at 2 min intervals. After a total incubationperiod of 14 min at 25° C., the virus was centrifuged and the titer wasdetermined. The control value of the preparation without Tween additionwas 10⁵.1. After the Tween treatment, the virus titer was below thedetection limit of 10⁰.5 and could be termed to be 0 based on thenegative tests with the reverse transcriptase. This results in a virusreduction factor of 7×5.1=35.7.

The solution freed from Tween was again admixed with HIV-1 virussuspension, lyophilized and, according to the method of EP-A-0 159 311heated at 60° C. for 10 h at a water content of 8%. The virus titer waslowered from 10⁶.2 to 0.

Both inactivation steps thus resulted in a total virus reduction factorof 41.9. Thus, it could be proven that a factor VIII preparation whichis subjected to the detergent and heat treatments under the conditionsstated above conforms to a total virus reduction factor of 41.9 and canbe viewed to be virus-safe.

The determination of the residual activity of factor VIII was effectedby the aid of the thromboplastin formation test (2 step test). Theresidual activity of factor VIII was calculated by forming the quotientof the factor VIII activity of the heated sample and the factor VIIIactivity of the starting material prior to the Tween treatment andamounted to 80%.

EXAMPLE 2

A preparation containing the clotting factors II, IX and X (partialprothrombin complex, PPC) was recovered according to the methoddisclosed in Vox. Sang. 33, 37-50 (1977) from human plasma by adsorptionon DEAE Sephadex, washing of the ionic exchanger and elution of thecomplex.

The PPC was admixed with HIV-1 in a solution containing 22% Tween 80 andincubated at 25° C. The virus suspension was added 15 times at 20 sintervals. The virus titer of the control without the addition of Tweenwas 10⁵.7. After the treatment with Tween the final virus titer wasbelow the detection limit of 10⁰.5. From this, a total virus reductionfactor of at least 15×5.2=78 is calculated. A PPC preparation subjectedto the above treatment with Tween thus corresponds to a total virusreduction factor of 78 and is to be viewed as virus-safe.

The activity of the coagulation factors was determined by way of factorIX via the addition of the sample to be tested to a factor IX deficientplasma and the determination of the activated partial thromboplastintime (1 step test) and was hardly influenced by the treatment withTween. The ratio of the activity of the treated sample treated to theactivity of factor IX in the untreated PPC was approximately 100%.

EXAMPLE 3

A PPC preparation as described in Example 2 was incubated at 25° C. with12% dimethyl octyl amine-N-oxide in the presence of model viruses(Sindbis and vesicular stomatitis virus=VSV). The addition of virussuspension was effected 10 times at 5 min intervals. After the treatmentwith detergent, the virus titer was below the detection limit of 10¹.5.The control values without detergent addition were 10⁶.4 and 10⁶.1,respectively. From this a total virus reduction factor of at least10×4.9=49 and 10×4.6=46 was calculated.

The biological activity was hardly impaired by the detergent treatmentand amounted to approximately 100%.

EXAMPLE 4

Plasma was fractionated according to Cohn, and the fibrinogen-containingCOHN I fraction was admixed with model viruses (Sindbis or VSV,respectively). After lyophilization, the concentrate having a watercontent of 8% was heated according to the method of EP-A-0 159 311 for10 hours at 60° C. and subsequently for 3 h at 80° C. The virus titerwas lowered by lyophilization from 10⁵.5 and 10⁶, respectively, to 10⁴.9and 10⁵.5, respectively, and furthermore, by the treatment at 60° C. tobelow the detection limit of 10⁰.5.

The inactivation capacity of the second treatment step at 80° C. wasdetermined in parallel preparations: lyophilization again lowered thevirus titer from 10⁵.5 and 10⁶.0 to 10⁴.9 and 10⁵.5, respectively, andfurther on, the treatment at 80° C. lowered it to below the detectionlimit.

The reduction factor is calculated from the two-time reduction by 5 or5.5 log-steps, respectivly, minus 0.6 or 0.5 log-steps, respectively,because during the two-step treatment the fibrinogen preparation wassubjected to a single lyophilization only. The reduction factors thuswere 9.4 and 10.5, respectively.

Subsequently, the powder was dissolved in a medium containing 5% octylglucoside, and at intervals of 5 min Sindbis or VSV, respectively, wasadded 9 times thereto. After the incubation (a total of 45 min at 25°C.) the virus titer was determined. The treatment with detergent reducedthe virus titer to a value of below the detection limit of 10⁰.5. Thecontrol value of a preparation without detergent addition was 10⁶.9 and10⁶.0, respectively. From this, virus reduction factors of at least 57.6and 49.5, respectively, were calculated. Thus, the total virus reductionfactors were at least 67.0 and 60.0, respectively. A fibrinogenpreparation which is subjected to the heat and detergent treatmentsunder the above indicated conditions conforms to a total virus reductionfactor of at least 60.0 and is to be considered as virus-safe.

A precipitate was precipitated by adding 8% ethyl alcohol to theoctyl-glucoside-containing fraction, and the biological activity of thefibrinogen was determined by means of a cross-linking test of thefibrin-α-chains (T. Seelich, H. Redl "Theoretische Grundlagen desFibrinklebers" in K. Schimpf "Fibrinogen, Fibrin und Fibrinkleber", F.K. Schattauer Verlag, Stuttgart-New York, 199-208, 1980) and by means ofthrombelastography (H. Hartert in "Thrombosis and Bleeding Disorders",(N. U. Bang et al., eds.) Georg Thieme Verlag Stuttgart, Acad. Press NewYork London, 70-76, 1971), coagulation factor XIII having been admixedin each case.

The biological activity of the treated fibrinogen, based on thebiological activity of the COHN I fraction, was 87%, measured by thecross-linking, and 56%, measured in the thrombelastogram.

EXAMPLE 5

Selected plasma was fractionated according to Cohn. The COHN IIIfraction which contained anti-tetanus-toxoid gamma globulin was admixedwith HIV-1 and Sindbis virus, respectively, in the presence of 15%Triton X-100 and incubated at 25° C. Virus addition was effected 30times at one-minute intervals. After an incubation period of a total of30 min, the virus titer was below the detection limit of 10².5 and10¹.5, respectively. The control values of the preparation without theaddition of detergent were 10⁵.7 and 10⁷.5, respectively. From this atotal virus reduction factor of at least 30×3.2=96 and 30×6=180,respectively, is calculated. A gamma globulin preparation which had beensubjected to the above detergent treatment corresponds to a total virusreduction factor of at least 96 and is to be viewed as virus-safe.

The biological activity was determined with an avidity test. For this,tetanus toxoid was adsorbed on a microtiter plate, covered with gelatineand washed. Subsequently, the gamma globulin to be tested was applied tothe coated microtiter plate in several dilutions, and non-adsorbedimmunoglobulin was washed off. The gamma globulin bound to the tetanustoxoid was determined by adsorption of an anti-human IgG peroxidaseconjugate to the Fc portion of the immunoglobulin and furthermore bycolor reaction of the peroxidase with diaminobenzidine and H₂ O₂ andsubsequent measurement of the optical density.

The avidity of the gamma globulin for the tetanus toxoid was hardlyinfluenced by the treatment with detergent. No significant aviditydifferences could be detected before and after the treatment.

EXAMPLE 6

0.95 ml of a solution containing C₁ esterase inhibitor (producedaccording to Vogelaar E F et al. (1973) Vox Sang. 26, 118-127"Contributions to the Optimal Use of Human Blood") were admixed with 20mg Triton X-100 and incubated at 25° C. To determine the virusinactivation capacity, VSV virus (10 μl) was repeatedly admixed atintervals of 5 min. When virus had been admixed 5 times, the virus titerwas detected to be below the detection limit of 10⁰.5. The control valueof the preparation without addition of detergent was 10⁷.5. From this, avirus reduction factor of at least 5×7=35 is calculated.

The C₁ esterase inhibitor was adsorbed on DEAE Sephadex and washed with8.89 g/l NaCl solution until it was free of detergent. After desorptionof the inhibitor with 59 g/l NaCl solution, it was dialysed against abuffer containing 1.0 g/l sodium citrate and 0.4 g/l NaCl (pH 6.8). Tothis solution again VSV virus was admixed before it was lyophilized. Thepreparation was dry-heated for 24 h at 72° C. During lyophilization andthe subsequent heat treatment, the virus titer was reduced from 10⁷.2 tobelow the detection limit of 10⁰.5. The virus reduction factor thus wasat least 6.7.

From the virus reduction factors of the detergent and thermaltreatments, a total virus reduction factor of at least 41.7 iscalculated. The biologic activity of the inhibitor was hardly impairedby the inactivation of the VSV viruses and was approximately 100%.

What is claimed is:
 1. A virally-inactivated blood product, other than an albumin product, that has a total virus reduction factor of at least 40 and that retains at least 50% of its pre-treatment biological activity, obtainable by an inactivation treatment whereina) a blood product is treated in an aqueous solution containing more than 10% by weight of a detergent without toxic solvents, and subsequently is heated while in a solid state to obtain the virally-inactivated blood product, or b) a blood product is heated while in a solid state and subsequently is treated in an aqueous solution containing more than 10% by weight of a detergent without toxic solve its to obtain the virally-inactivated blood product, wherein the virally-inactivated blood product obtainable by said inactivation treatment has a detergent content but is free of toxic solvents.
 2. A blood product according to claim 1, wherein said solid state has a water, methanol or ethanol content of 5% by weight to 70% by weight.
 3. A blood product according to claim 1, wherein the heating of said blood product is in a closed container at a temperature range of 50° C. to 121° C.
 4. A method of producing a virally-inactivated blood product having a detergent content but free of toxic solvents, other than an albumin product, comprisirg the steps of, in any order:treating a blood product in an aqueous solution containing more than 10% by weight of a detergent without toxic solvents, and heating the blood product in a solid state to obtain the virally-inactivated blood product, whereby a total virus reduction factor of at least 40 is achieved and the virally-inactivated blood product retains at least 50% of its pre-treatment biological activity.
 5. A method according to claim 4, wherein said solid state has a water, methanol or ethanol content of from 5% by weight to 70% by weight.
 6. A method according to claim 4, wherein the heating of said blood product is in a closed container at a temperature range of 50° C. to 121° C.
 7. A method of inactivating viruses in a blood sample to produce a non-albumin, virally-inactivated blood product having a detergent content but free of toxic solvents, comprising the steps of, in any order:treating said blood sample in an aqueous solution with a detergent present in an amount of at least 2% by weight without toxic solvents; and heating said blood sample in a solid state, wherein said blood sample has a water, methanol or ethanol content of 5% to 70% by weight and is not in solution, whereby after said treating and heating steps said virally-inactivated blood product retains at least 50% of its pre-treatment biological activity and wherein said virally-inactivated blood product has a virus reduction factor of at least
 40. 8. A method according to claim 7, wherein said blood product is a coagulation factor.
 9. A method according to claim 7, wherein said blood product is an immunoglobulin. 